Apoptosis Inducing Factor Acts as a Survival Factor in Acute Myelogenous Leukemia through Mitochondrial Complex 1.

Apoptosis inducing factor (AIF) is a mitochondrial flavoprotein that is released into the cytoplasm to form an active DNase and then translocates into the nucleus to induce DNA fragmentation when cells undergo apoptosis. AIF also acts as an essential NADPH oxidase, a function which is distinct from its proapoptotic role. We have shown previously that AIF expression level increased with increasing blast counts in primary acute myelogenous leukemia (AML) patient samples. Here we report that AIF expression levels are increased in primary CD34⁺ AML cells compared with CD34⁻ cells. To investigate the role of AIF in primary AML, bone marrow or peripheral blood samples from AML patients (n=8) with CD34⁺ or CD34⁻ cell populations were examined for changes in reactive oxygen species (ROS), mitochondrial membrane potential (ΔψM), apoptosis and chemo-sensitivity in the CD34+ and CD34− compartments after treatment with the mitochondrial complex 1 inhibitor rotenone. Similar experiments were performed following knockdown of AIF with lentiviral shRNA directed against AIF in K562 cells.

Results demonstrate:

1. significantly increased apoptosis rate (39.2% Annexin V+ cells in CD34⁺ vs. 14.1% in CD34− cells following 2 uM rotenone, p=0.05), and decreased ΔψM in CD34⁺ cells (44.5% ΔψM cells in CD34⁺ vs. 78.2% in CD34⁻ cells, p=0.01). MCL-1 expression levels were significantly decreased in CD34⁺ but not in CD34⁻ cells (p=0.007). The apoptotic effect of rotenone in CD34⁺ cells was potentiated by AraC (1uM).

2. Profound inhibition of O₂ consumption in AIF knockdown K562 cells (−5.2±0.3) compared with K562-neo (neo transfected K562, −13.1±0.3) cells (p=0.001).

Rotenone treated AIF knockdown K562 cells and K562-neo cells revealed that decreased AIF expression led to higher ΔψM and higher superoxide generation (p=0.0002). In contrast, total mitochondrial mass (NAO) decreased slightly in K562KD cells. Data suggested that

1. AIF contributes to leukemia cell survival by modulating mitochondrial metabolism, independent of its proapoptotic activity, which is consistent with recent reports demonstrating its importance in mitochondrial complex 1 activity.

2. Decreased AIF levels are associated with increased generation of superoxide anion, suggesting that AIF critically regulates mitochondrial function in AML cells.

3. Rotenone selectively decreases AIF and induces apoptosis in CD34⁺ cells in AML.

4. AIF functions as a survival factor in AML strategies targeting AIF expression or function may be beneficial in the treatment of myeloid leukemia.